Biotechnology: Principles and Processes — Questions & Answers

Sample questions from Biotechnology: Principles and Processes

1. Q1. The specific DNA sequence that a restriction endonuclease recognizes and binds to before cutting is called a:

   • A.Promoter
   • B.Recognition sequence (recognition site)✓
   • C.Origin of replication
   • D.Terminator
   Solution

   The recognition sequence is the specific, often palindromic, DNA sequence that a particular restriction endonuclease specifically recognizes and binds to before cleaving the DNA at that site.

2. Q2. During downstream processing, suitable preservatives are added to the product at the stage of:

   • A.Formulation✓
   • B.Separation
   • C.Cell lysis
   • D.Sparging
   Solution

   Downstream processing consists of separation and purification of the product followed by formulation with suitable preservatives, which extends shelf life and maintains activity. Formulation precedes clinical trials and quality control for drug products.

3. Q3. Blunt-ended fragments generated by SmaI are ligated into a SmaI-cut vector, and the yield of recombinants is far lower than in a comparable sticky-end ligation. The reason is:

   • A.Blunt ends offer no complementary overhang to hold the two molecules together, so ligase has fewer correctly juxtaposed ends to seal✓
   • B.DNA ligase cannot form phosphodiester bonds between blunt ends at all
   • C.SmaI methylates the ends it produces, blocking ligation
   • D.Blunt ends carry no phosphate groups, so no bond is possible
   Solution

   Sticky ends base-pair transiently and hold the partners in register while ligase seals the nicks, which raises the effective efficiency. Blunt ligation works, but depends on chance collisions, so recombinant yield falls; the insert can also enter in either orientation.

4. Q4. Agrobacterium tumefaciens and retroviruses are both described as disarmed vectors. What exactly is removed or altered when they are disarmed?

   • A.The origin of replication, so that they cannot multiply in the host
   • B.The single-stranded genome, which is replaced by double-stranded DNA
   • C.Their ability to enter host cells, which is replaced by chemical uptake
   • D.The genes responsible for causing disease in the host, while the machinery that delivers DNA into the cell is retained✓
   Solution

   Both are natural gene-delivery systems: Agrobacterium transfers T-DNA to plant cells and retroviruses can transform animal cells. Disarming deletes the pathogenic or tumour-inducing functions while preserving the transfer apparatus, so a desirable gene is delivered without causing disease.

5. Q5. In pBR322 the PstI and PvuI sites lie inside ampR while BamHI and SalI lie inside tetR. A clone made by inserting DNA at PstI is plated on tetracycline and, separately, on ampicillin. The expected result is:

   • A.Growth on ampicillin but not on tetracycline
   • B.Growth on both antibiotics
   • C.Growth on tetracycline but not on ampicillin✓
   • D.No growth on either antibiotic
   Solution

   The insert at PstI splits the ampicillin resistance gene, so beta-lactamase is not made and the cells are ampicillin sensitive. The tetracycline resistance gene is untouched, so the recombinants grow on tetracycline. Reversing the site to BamHI would reverse the two phenotypes.

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